Steven W. Levison, PhD - US grants

Previous studies on the rodent forebrain have defined a single precursor cell whose progeny can differentiate into two glial classes previously thought to arise from separate progenitors. Preliminary studies suggest that this precursor also has the potential to produce neurons as well as both glial lineages. The goal of this grant will be to determine the relative contributions of cell lineage and local environmental cues in shaping the fate of this cell's progeny. To establish the role of lineage in regulating fate, the following two hypotheses shall be tested: 1) that this precursor is genetically determined to divide asymmetrically to generate astroblasts and O-2A progenitors; and 2) that programmed cell death regulates the timing of postnatal gliogenesis by eliminating cells that develop inappropriately. To determine the role of cell-cell interactions in regulating cell fate, co-culture experiments will be performed with astrocytes, oligodendrocytes, neurons, and endothelial cells to test the hypothesis that contact with, or secreted growth factors from, these cells affect the differentiation of the multipotential precursor. To determine whether the same precursor that produces both classes of glia can also produce neurons, the multipotential glial precursor will be purified using immunoselection and grown in culture medium that fosters neuronal growth. A clonal analysis will establish whether this progenitor is pluripotent. Finally, the purified progenitor cell will be transplanted into fetal or juvenile hosts as another assay of its developmental potential. The outcome of these studies will profoundly influence current views on the development of the mammalian cerebral cortex. These studies will provide insights into the cellular origins of glial brain tumors and future characterizations of this precursor may provide insights that will assist the medical profession in treating individuals who suffer from neurological diseases and traumatic injuries to the nervous system.

The overall goal of this proposal is to establish how perinatal hypoxia/ischemia (H/I) affects the neural stem cells in the subependymal zone (SZ). The SC cells are actively dividing during the perinatal period to generate neurons and glia. Therefore, insults that destroy or disturb SZ cells will likely disrupt brain development and function. In the last 8 years my collaborators and I have collected a wealth of data on the normal development fates of these stem cells. Based on our extensive characterization, a number of predictions can be made as to how a H/I insult during this critical period of brain development will affect these progenitors. Specific aim 1 will investigate the prediction that H/I will alter the rate or number of proliferating cells in the SC. Specific aim 2 will determine whether H/I kills cells in the SZ. Experiments will be performed to establish the proportion of necrotic versus apoptotic deaths and to reveal the mechanisms responsible for their demise. Since each progenitor has the potential to produce over 100 daughters, the death of even a small number of proliferating stem cells could have a dramatic impact on brain development. Specific aim 3 will establish whether H/I will disrupt the rate or pattern of migration of progenitors from the SZ. A H/I insult will disturb or destroy the radial glial network that immature cells use for migration, and this will likely alter the precise timing or direction of cellular movements that occur during normal brain development. Finally, specific aim 4 will determine whether there is abnormal cell differentiation in the wake of a H/I insult. By altering the micro-environment of the developing brain, perinatal insults also will change the signals that determine cell identify. Our preliminary data indicate that H/I insults impact all of the parameters discussed above. Therefore, completion of these experiments should provide insight into the mechanisms whereby the perinatal hum brain is adversely influence by even mild H/I insults.

The overall goal of this proposal is to establish how perinatal hypoxia/ischemia (H/I) affects the neural stem cells in the subependymal zone (SZ). SZ cells divide constitutively during the perinatal period to generate neurons and glia. Insults that destroy or disturb SZ cells, therefore, will likely disrupt brain development and function. In the last 8 years my collaborators and I have collected a wealth of data on the normal developmental fates of these stem cells. Based on our extensive characterization a number of predictions can be made as to how a H/I insult during this critical period of brain development will affect these progenitors. Specific aim 1 will investigate the prediction that H/I will alter the rate or number of proliferating cells in the SZ. Specific aim 2 will determine whether H/I kills cells in the SZ. Experiments will be performed to establish the proportion of necrotic vs apoptotic deaths and to reveal the mechanisms responsible for their demise. Since each progenitor has the potential to produce over 100 daughters, the death of even a small number of proliferating stem cells could have a dramatic impact on brain development. Specific aim 3 will establish whether H/I will disrupt the rate or pattern of migration of progenitors from the SZ. A H/I insult will disturb or destroy the radial glial network that immature cells use for migration, and this will likely alter the precise timing or direction of cellular movements that occur during normal brain development. Finally, Specific aim 4 will determine whether there is abnormal cell differentiation in the wake of a H/I insult. By altering the microenvironment of the developing brain, perinatal insults also will change the signals that determine cell identity. While periventricular hemorrhage is a frequent consequence of perinatal asphyxia of the newborn, the consequences of this injury have not been closely examined or considered important. Our preliminary data indicate that H/I insults impact all of the parameters discussed above. Completion of these experiments will reveal how damage to the subependymal zone contributes to hypoxic/ischemic damage in the perinatal human infant.

DESCRIPTION (provided by applicant): We have recently established that there is an early regenerative response initiated by the neural stem cells (NSCs) in the subventricular zone (SVZ) in response to a perinatal hypoxic/ischemic (H/I) insult; therefore, the goals of this proposal are to establish the molecular mechanisms responsible for this adaptive change and to determine whether we can amplify this response. Preliminary data indicate that: 1) there is increased proliferation within the SVZ within the region that is known to contain the NSCs; 2) there is a transient increase in the abundance of multipotential NSCs after perinatal H/I; 3) NSCs placed in vitro retain a memory of the insult; and 4) these cellular changes are accompanied by changes in the expression of several ligands and receptors that have been implicated in stem cell maintenance. To our knowledge, these are the first available data demonstrating that there is a change in the numbers of multipotential NSCs as a consequence of any insult to the CNS. Consequently, the overall hypothesis of this application is that there is an increase in the number of stem cells in the H/I brain as a consequence of both intrinsic changes within the stem cells as well as extrinsic changes to their niche. The following specific aims are proposed: Aim 1: To determine the magnitude and kinetics of the stem cell response to perinatal H/I; Aim 2. To test the hypothesis that brain damage increases the frequency of symmetrical NSC amplifying cell divisions; Aim 3: To identify the molecules that amplify neural stem cell numbers after perinatal H/I; Aim 4: To determine whether LIF or Notch1 receptors regulate NSC numbers after H/I; Aim 5. To determine whether the increased abundance of neural stem cells is a consequence of a direct effect on the cells or whether this increase is a result of changes to the stem cell niche. As there is great interest in exploiting stem cells for CNS regeneration, completion of the proposed studies will enhance our fundamental understanding of the signals that maintain these precursors as neural stem cells and which signals regulate their abundance in vivo. It must be emphasized that at the present time, there are no therapies available to protect the infant brain from perinatal insults or stimulate regeneration. Moreover, as it is likely that the majority of perinatal insults occur during the antenatal period and that such events occur undetected, the knowledge obtained from these studies could eventually lead to therapeutics that could be administered to infants to enable the damaged infant brain to develop normally from its endogenous stem cells. Such therapies would clearly have advantages over transplantation approaches, and if effective would decrease the incidence and life long cognitive, motor and emotional handicaps that occur as a result of perinatal brain damage.

DESCRIPTION (provided by applicant): There are a number of neurological disorders that result from perinatal brain damage. While there is no single cause of these disorders, there is a strong correlation between hypoxia-ischemia (H/I) and damage to the cerebral gray and white matter. Despite progress in understanding the pathogenesis of hypoxic-ischemic injury, we have an incomplete appreciation for the mechanisms leading to permanent brain injury and more importantly there is no clear explanation for the failure of regeneration. Increasingly, investigators are beginning to appreciate the impact of this injury on the subventricular zone (SVZ), which is that region of the immature brain that harbors the multipotential neural stem cells/progenitors (NSPs) that are endowed with the ability to generate neurons, astrocytes and oligodendrocytes. The specific goal of this proposal is to identify signals that induce the differentiation of glial precursors towards astrocytes and to evaluate recovery from H/I when specific astrocyte inducers are antagonized. Our preliminary data indicate that there is an increase in the production of astrocytes from SVZ cells at the expense of myelinating oligodendrocytes following recovery from perinatal H/I. Thus, our hypothesis is that as a result of injury-induced cytokines, that there is aberrant production of astrocytes from glial progenitors in the SVZ and that this glial dysgnesis contributes to the permanent deficit in white matter oligodendrocytes that occurs subsequent to H/I. Seminal studies on brain development have shown that neural precursors are responsive to extrinsic signals that govern their differentiation choices. We hypothesize that as a result of an H/I insult that specific signals are now present that would otherwise be absent. The specific aims of this proposal are to: 1) Test the hypothesis that perinatal H/I increases the production of astrocytes from glial progenitors in the SVZ; 2) Test the hypothesis that the damaged neonatal brain produces factors that promote astrocyte generatation from postnatal SVZ cells; 3) Establish which transcription factors are active in postnatal SVZ cells by relevant astroglial inducers; and 4) Establish whether a pharmacological antagonist of the TGF-¿ receptor, ALK5, will prevent the aberrant production of astrocytes in vitro and in vivo after H/I. We anticipate that the knowledge obtained from these studies will lead to pharmacological interventions for infants surviving H/I or other disturbances of brain development to enable the infant brain to develop more normally. The insights we gain from our studies also will be applicable to a broad range of injuries and diseases of the CNS, as well as to studies where exogenous neural precursors will be transplanted into the brain, where excessive astroglial differentiation or astrogliosis are deemed undesirable. PUBLIC HEALTH RELEVANCE: Despite progress in understanding the pathogenesis of hypoxic-ischemic injury, we have an incomplete appreciation for the mechanisms leading to permanent brain injury and more importantly there is no clear explanation for the failure of regeneration. The specific goal of this proposal is to identify signals that induce the differentiation of neural precursors towards astrocytes and to evaluate recovery from H/I when specific astrocyte inducers are antagonized. The insights we gain from our studies also will be applicable to a broad range of injuries and diseases of the CNS, as well as to studies where exogenous neural precursors will be transplanted into the brain, where excessive astroglial differentiation or astrogliosis are deemed undesirable.

DESCRIPTION (provided by applicant): The current application is for support for the 43rd annual ASN meeting to be held in Baltimore, MD from March 3rd to 7th, 2012. NIH funding for previous ASN meetings has been invaluable for supporting our scientific programs and for enhancing our ability to involve graduate students and postdoctoral researchers in the ASN meeting. To accommodate the breadth of neurochemistry as well as cellular and molecular neurobiology and to provide in depth analyses of particular topics, the ASN continues to build its scientific program around four interwoven, but distinct, themes. These themes have been selected to increase our understanding of the cellular and molecular bases of neural development and disease. These themes are: Building the Nervous System. Metabolism and Cell & Molecular Neuroscience. Glial Mechanisms & Injury. Neuronal Degeneration & Disease. The ASN meeting provides numerous opportunities for delegates to exchange ideas and to form new collaborations because total attendance is approximately 500 and there are numerous spaces in our program for delegates to congregate informally. The Society also has several mechanisms to enhance the professional development of junior investigators during the meeting. We organize luncheons with the plenary speakers, which gives attendees a chance to discuss topics with the speakers. The fees for these luncheons are reduced for students and postdoctoral fellows. There is a dinner exclusively for students and postdoctoral fellows, which is repeatedly recognized as an outstanding opportunity to network. There is a luncheon entitled Women in Neurochemistry, that is open to all attendees, and junior investigators have reduced fees. There are up to 6 sessions that are selected from abstracts submitted, with an emphasis on choosing presentations from graduate students and post doctoral fellows. There are travel awards for outstanding graduate students and post doctoral fellows to defray their costs of attendance. We host a job posting site and students can meet with potential future mentors or colleagues during the meeting. Most recently, we added a half-day program for visiting high-school students to engage these future scientists during their formative years. From results of yearly exit surveys, we know that the annual ASN meeting has and will continue to provide an excellent venue for cutting edge neurochemistry and for enhancing the careers of young investigators. PUBLIC HEALTH RELEVANCE: This proposal requests support for the 43rd annual meeting of the American Society for Neurochemistry taking place March 3rd to 7th in St Louis, MO. Support for this meeting will provide valuable assistance to this Society to publicly present new developments in neurochemistry. This annual meeting provides the latest information on advances in molecular and cellular neurobiology. Studies on numerous neurological diseases are presented. A Public Forum is held, which is advertised and focused on presenting information to the general public, this year on traumatic brain injury. A large part of the support requested will help keep registration fees low for young investigators and be used to support educational and networking events for high school students, graduate students, postdocs, young investigators, women and minorities.

DESCRIPTION (provided by applicant): The current application is for support for the 44th annual ASN meeting, which will be held as a joint meeting with the International Society for Neurochemistry in Cancun, Mexico April 20th-24th, 2013. NIH funding for previous ASN meetings has provided invaluable support for our scientific programs and for enhancing graduate student and postdoctoral fellow attendance and involvement in our meeting. NIH support this year will not only help ASN achieve its goals, but will assist the US Government in achieving its goals of promoting the establishment of global scientific networks and in enhancing diplomatic relations in countries where relationships are strained. With this year's meeting joined with the annual ISN meeting, there will be even greater breadth to our scientific conference ensuring a richer intellectual experience for meeting delegates. This year's ASN meeting will provide increased opportunities for delegates to exchange ideas and to form new international collaborations because total attendance is expected to be 1300 scientists from over 30 countries. While the scientific program is rich, there will be numerous spaces in our program for delegates to congregate informally during the meeting or at pre-meeting satellites. The Society will continue its tradition of enhancing the professional development of junior investigators during the meeting through several mechanisms. We have organized luncheons with our internationally recognized plenary speakers to give our fellows opportunities to informally meet with these speakers. The fees for these luncheons are reduced for students and postdoctoral fellows. There is a dinner exclusively for students and postdoctoral fellows, which is repeatedly recognized as an outstanding opportunity to network. There is a luncheon entitled Women in Neurochemistry that is open to all attendees, and junior investigators have reduced fees. There are 3, 30 minute featured lectures for young investigators and 3 sessions that are selected from abstracts submitted that will allow up to 18 fellows to present their research orally. There are travel awards for outstanding graduate students and postdoctoral fellows to defray their costs of attendance. We host a job- posting site and students can meet with potential future mentors or colleagues during the meeting. Recently, we added a half-day program for visiting high- school students to engage these future scientists during their formative years, (which we are introducing to the ISN at this meeting) to bring talented Mexican high school students to our conference. From results of yearly exit surveys, we know that the annual ASN meeting has and will continue to provide an excellent venue for cutting edge neuroscience and for enhancing the careers of young investigators.

DESCRIPTION (provided by applicant): Neural precursors have long been of interest to developmental biologists. These cells have recently gained the interest of the broader neuroscience community because of their involvement in olfaction, learning and memory, cognitive decline with aging and their potential to replace neurons or glial cells that have died a a consequence of brain injury or disease. The cells that are of significant interest are the neural stem cells (NSCs). These cells naturally reside within specific niches where they receive signals that are necessary to maintain them in a primitive state. To date, the possibility that IGF-II is a necessary component of the stem cell niche has not been considered largely because IGF-II has been regarded as a fetal growth factor. However, IGF-II is expressed at high levels within the choroid plexus, which produces the cerebrospinal fluid that is readily accessible to the NSCs because they extend a process directly into the ventricle that is bathed by cerebrospinal fluid. Whereas both IGF-II and IGF-I activate the IGF type 1 receptor (IGF-1R), IGF-II also binds to a splice variant isoform of the insulin receptor (IR-A) supporting distinct roles for IGF-II versus IGF-I. The overall hypothesis of this proposal is that IGF-II is essential for NSC self-renewal, maintenance and growth through the insulin receptor. Our overall hypothesis will be tested using inducible Cre driver lines to achieve both temporal discrete deletion of IGF-II or insulin receptors. Studies will be performed at the molecular, cellular and behavioral levels. Identifying IGF-II as necessary to sustain NSCs as primitive cells will be a significant scientific advance. Moreover, establishing which signaling receptor and downstream transcription factors are activated by this signal might well provide insights into new strategies to amplify these important cells to promote brain growth, maintain cell replacement across the lifespan and enhance cell replacement in the diseased or damaged brain. IGF-II is also expressed in other organs where there are adult stem cells, yet a role for IGF-II in adult stem cell maintenance has not been explored in mammalian tissues. Therefore, upon completing the proposed experiments we will be uniquely positioned to submit an R01 application to investigate these important and timely issues.

Abstract The subventricular zone (SVZ) contains neural stem cells and progenitors of various potentialities. Although initially parsed into A, B, and C cells, it is clear that this germinal zone is comprised of a significantly more diverse population of cells. We have established a flow cytometry panel that has revealed the existence of neural stem cells as well as 4 types of multipotential transit amplifying progenitors (TAPs) and 3 types of glial restricted TAPs. One of these TAPs, the platelet-derived growth factor (PDGF) and fibroblast growth factor-responsive multipotential progenitor (PFMP) expresses the PDGF? receptor and proliferates in response to PDGF. The other PDGFR?+ cell is a bipotential glial restricted TAP (GRP3). The central premise of this application is that the PDGF-responsive progenitors (PRPs) represent an important subset about which little is known. We provide preliminary data that suggest that these PRPs are descended from the lateral ganglionic eminence and that their proliferation can be positively and negatively affected by environmental signals. Therefore, the goal of this exploratory R21 is to: 1) Establish when the PFMPs and GRP3s emerge during mouse development; 2) Define the transcripts expressed by individual PRPs using the dropseq method and then use the Monocle algorithm to identify unique subpopulations. We will then proceed to compare and contrast the transcriptional profiles of the PRP subgroups to each other and to the stem cells (using both our data, and publicly available data) to define changes in gene transcription that correspond with their developmental restriction and specification; and 3) Establish which more restricted progenitors are descended from the PFMPs and define the types of neurons and glia that they produce in vivo. These formative studies will lay the foundation for a decade of future work to understand how these progenitors participate in neural development across species, how they are affected by environmental stimuli that disturb normal brain development leading to brain disorders and how these progenitors might be recruited to participate in regeneration and repair of the nervous system after injury.

Our studies have established that there is an early regenerative response initiated by the neural stem cells and progenitors (NSPs) in the subventricular zone (SVZ) in response to a perinatal hypoxic-ischemic (H-I) insult and that the expansion of the NSPs requires the cytokine leukemia inhibitor factor (LIF). We have collected new data that reveals other functions of LIF as LIF haplodeficient animals sustain worse injury compared to wild type mice. Complementing those loss of function studies, we provide preliminary data to show that delayed intranasal LIF administration, reduces the extent of cerebral neuronal loss, increases proliferation in the SVZ and improves neurological function in a mouse model of near term hypoxia-ischemia. Therefore, the premise of this proposal is that LIF is an essential neuroprotective and regenerative cytokine and that the non-invasive, intranasal administration of LIF can promote regeneration and decrease the long-term burden of neurological deficits. We will test this premise by performing experiments using pre-term and near-term mouse models of perinatal injury with the following 3 specific aims: 1) that LIF haplodeficient mice will sustain greater neuronal and glial cell damage after a developmental brain injury accompanied by worse neurological disabilities; 2) that delayed intranasal LIF administration will stimulate the numbers of stem cells and progenitors to repair the damaged gray and white matter and 3) that the extent of axonal regeneration and function can be improved by delayed intranasal LIF administration. During the course of our studies we will elucidate the mechanisms through which LIF is exerting its potent neuroprotective and regenerative actions. As it is likely that many perinatal insults occur during the antenatal period and go undetected, the knowledge obtained from these studies could lead to therapeutics that could be administered to infants subacutely to enable the damaged brain to develop normally from its endogenous stem cells, thus decreasing the incidence and life long cognitive, motor and emotional handicaps that occur as a result of developmental brain damage.